Gemcitabine combination therapy

ABSTRACT

The present invention provides compositions and methods for the treatment of cell proliferative disorders using at least one DAC inhibitor and gemcitabine.

BACKGROUND OF THE INVENTION

Dysregulation or loss of control of cell division can result in the development of any of a variety of cell proliferative disorders, many of which are debilitating or deadly. Although much has been learned about mechanisms involved in cell proliferation, and therefore about common biological principles underlying a variety of different disorders, there remains a need for the development of new and/or improved therapies for the treatment of such conditions.

There is a particular need for the development of improved therapies for the treatment of tumors that express the Ras oncogene. Ras-expressing tumors are often more resistant to standard therapies. Furthermore, many of the most deadly cancers involve Ras-expressing tumors. For example, 90-95% of pancreatic tumors are Ras-expressing. Similarly, 40-45% of colorectal tumors, 40% of bladder tumors, 15-20% of non small cell lung carcinomas express Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS), which are not themselves cancer but are bone marrow disorders characterized by abnormal cell maturation that typically progress to cancer (AML), also express Ras. There is a profound need for the development of therapies for these and other Ras-expressing diseases and disorders.

SUMMARY OF THE INVENTION

The present invention encompasses the finding that combinations of DAC inhibitors with gemcitabine are have particular utility in the treatment of proliferative diseases. Among other things, the invention establishes the particular utility of DAC inhibitor/gemcitabine combination therapy in treatment of tumors expressing the Ras oncogene. In certain particular embodiments, combination therapy with romidepsin and gemcitabine is provided, for example for use in the treatment of proliferative disorders generally and/or for use in the treatment of tumors expressing the Ras oncogene.

The present invention provides methods of treating a proliferative disorder by administering a combination of one or more DAC inhibitors and gemcitabine.

The present invention further provides methods of treating tumors that express the Ras oncogene by administering a DAC inhibitor together with gemcitabine. In some embodiments, such methods involve determining that a tumor expresses the Ras oncogene, and then, administering combination therapy with a DAC inhibitor and gemcitabine. Determination that a tumor expresses the Ras oncogene can involve testing for expression of the Ras oncogene and/or can involve determining that the tumor is of a type that typically expresses the Ras oncogene.

The present invention provides combination regimens, and unit dosages of pharmaceutical compositions useful in such regimens. The present invention further provides kits for combination therapy of DAC inhibitors and gemcitabine.

DESCRIPTION OF THE DRAWING

FIGS. 1-3 depict structures of certain DAC inhibitors that, like other DAC inhibitors available in the art and/or described herein, may be utilized in some embodiments of the present invention.

FIG. 4 shows the effects of depsipeptide (FK228) alone and in combination with gemcitabine in in vivo mouse xenograft model of Ras-expressing pancreatic tumor.

DEFINITIONS

Alicyclic: The term “alicyclic,” as used herein, denotes a monovalent group derived from a monocyclic or bicyclic saturated carbocyclic ring compound by the removal of a single hydrogen atom. Examples include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Such alicyclic groups may be further substituted.

Aliphatic: An “aliphatic group” is non-aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted.

Aryl: The term “aryl,” as used herein, refers to a mono- or polycyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.

Cell Proliferative Disorder, Disease, or Condition: The term “cell proliferative disease or condition” is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation.

Combination Therapy: According to the present invention, a DAC inhibitor may desirably be administered in combination with gemcitabine. Such therapy will commonly involve administration of multiple individual doses of a DAC inhibitor and/or of gemcitabine, spaced out over time. Doses of a DAC inhibitor and gemcitabine may be administered in the same amounts and/or according to the same schedule or alternatively may be administered in different amounts and/or according to different schedules.

DAC Inhibitor: In general, any agent that specifically inhibits a deacetylase is considered to be a DAC inhibitor. Any agent that specifically inhibits a histone deacetylase is considered to be an HDAC inhibitor. Those of ordinary skill in the art will appreciate that, unless otherwise set forth herein or known in the art, DAC inhibitors may be administered in any form such as, for example, salts, esters, prodrugs, metabolites, etc. Furthermore, DAC inhibitors that contain chiral centers may be administered as single stereoisomers or as mixtures, including racemic mixtures, so long as the single stereoisomer or mixture has DAC inhibitor activity.

DAC Inhibitor Therapy: As used herein, the phrase “DAC inhibitor therapy” refers to the regimen by which a DAC inhibitor is administered to an individual. Commonly, DAC inhibitor therapy will involve administration of multiple individual doses of a DAC inhibitor, spaced out over time. Such individual doses may be of different amounts or of the same amount. Furthermore, those of ordinary skill in the art will readily appreciate that different dosing regimens (e.g., number of doses, amount(s) of doses, spacing of doses) are typically employed with different DAC inhibitors.

Electrolyte: In general, the term “electrolyte”, as used herein, refers to physiologically relevant free ions. Representative such free ions include, but are not limited to sodium(Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), chloride (Cl⁻), phosphate (PO4³⁻), and bicarbonate (HCO₃ ⁻).

Electrolyte Supplementation: The term “electrolyte supplementation”, as used herein, refers to administration to a subject of a composition comprising one or more electrolytes in order to increase serum electrolyte levels in the subject. For purposes of the present invention, when electrolyte supplementation is administered “prior to, during, or after” combination therapy, it may be administered prior to initiation of combination therapy inhibitor therapy (i.e., prior to administration of any dose) or prior to, concurrently with, or after any particular dose or doses.

Halogen: The term “halogen”, as used herein, refers to an atom selected from fluorine, chlorine, bromine, and iodine.

Heteroaryl: The term “heteroaryl”, as used herein, refers to a mono- or polycyclic (e.g. bi-, or tri-cyclic or more) aromatic radical or ring having from five to ten ring atoms of which one or more ring atom is selected from, for example, S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

Heterocyclic: The term “heterocyclic”, as used herein, refers to a non-aromatic 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (iv) any of the above rings may be fused to a benzene ring, and (v) the remaining ring atoms are carbon atoms which may be optionally oxo-substituted. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and tetrahydrofuryl. Such heterocyclic groups may be further substituted.

Initiation: As used herein, the term “initiation” when applied to therapy can refer to a first administration of an active agent (e.g., a DAC inhibitor or gemcitabine) inhibitor to a patient who has not previously received a DAC inhibitor. Alternatively or additionally, the term “initiation” can refer to administration of a particular dose of a DAC inhibitor and/or of gemcitabine during therapy of a patient.

Pharmaceutically acceptable carrier or excipient: As used herein, the term “pharmaceutically acceptable carrier or excipient” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

Pharmaceutically acceptable ester: As used herein, the term “pharmaceutically acceptable ester” refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.

Pharmaceutically acceptable prodrug: The term “pharmaceutically acceptable prodrugs” as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention. “Prodrug”, as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of the invention. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). “Design and Application of Prodrugs, Textbook of Drug Design and Development”, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

Pharmaceutically acceptable salt: As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable include, but are not limited to, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

Stable: The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject). In general, combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.

Substituted: The terms “substituted aryl”, “substituted heteroaryl”, or “substituted aliphatic,” as used herein, refer to aryl, heteroaryl, aliphatic groups as previously defined, substituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to, —F, —Cl, —Br, —I, —OH, protected hydroxyl, —NO₂, —CN, —C₁-C₁₂-alkyl optionally substituted with, for example, halogen, C₂-C₁₂-alkenyl optionally substituted with, for example, halogen, —C₂-C₁₂-alkynyl optionally substituted with, for example, halogen, protected amino, —NH—C₁-C₁₂-alkyl, —NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkenyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl, dialkylamino, -diarylamino, -diheteroarylamino, —O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl, —O—C₂-C₁₂-alkenyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₂-C₁₂-alkenyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkenyl, —CONH—C₃-C₁₂-cycloalkyl, —CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkenyl, —OCONH—C₃-C₁₂-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl, —NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl, —NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₂-C₁₂-alkenyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂— aryl, —NHCO₂— heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂, —NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₂-C₁₂-alkenyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl, —NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂, —NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₂-C₁₂-alkenyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl, —NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂, —NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₂-C₁₂-alkenyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl, —NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl, —NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₃-C₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl, —NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl, —C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl, —C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl, S(O)—C₂-C₁₂-alkenyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₃-C₁₂-cycloalkyl, —SO₂NH— aryl, —SO₂NH-heteroaryl, —SO₂NH— heterocycloalkyl, —NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl, —NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH, —S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, or methylthiomethyl. It is understood that the aryls, heteroaryls, alkyls, and the like can be further substituted.

Susceptible to: The term “susceptible to”, as used herein refers to an individual having higher risk (typically based on genetic predisposition, environmental factors, personal history, or combinations thereof) of developing a particular disease or disorder, or symptoms thereof, than is observed in the general population.

Therapeutically effective amount: The term “therapeutically effective amount” of an active agent or combination of agents is intended to refer to an amount of agent(s) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of a particular agent may range from about 0.1 mg/Kg to about 500 mg/Kg, preferably from about 1 to about 50 mg/Kg. Effective doses may also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of any particular active agent utilized in accordance with the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.

Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a biologically active agent that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

As indicated, the present invention demonstrates that combinations of DAC inhibitors and gemcitabine are particularly useful in the treatment of proliferative disorders.

Cell Proliferative Disorders, Diseases, or Conditions

In some embodiments, the invention provides methods for treating cell proliferative disorders, diseases or conditions. In general, cell proliferative disorders, diseases or conditions encompass a variety of conditions characterized by aberrant cell growth, preferably abnormally increased cellular proliferation. For example, cell proliferative disorders, diseases, or conditions include, but are not limited to, cancer, immune-mediated responses and diseases (e.g., transplant rejection, graft vs host disease, immune reaction to gene therapy, autoimmune diseases, pathogen-induced immune dysregulation, etc.), certain circulatory diseases, and certain neurodegenerative diseases.

In certain embodiments, the invention relates to methods of treating cancer. In general, cancer is a group of diseases which are characterized by uncontrolled growth and spread of abnormal cells. Examples of such diseases are carcinomas, sarcomas, leukemias, lymphomas and the like.

For example, cancers include, but are not limited to leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndrome, mesothelioma, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular, rectal and colon), lung cancer, breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, liver cancer and thyroid cancer, and/or childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas.

In some embodiments, the invention relates to treatment of leukemias. For example, in some embodiments, the invention relates to treatment of chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, and/or adult T cell leukemia/lymphoma. In certain embodiments, the invention relates to the treatment of AML. In certain embodiments, the invention relates to the treatment of ALL. In certain embodiments, the invention relates to the treatment of CML. In certain embodiments, the invention relates to the treatment of CLL.

In some embodiments, the invention relates to treatment of lymphomas. For example, in some embodiments, the invention relates to treatment of Hodgkin's or non-Hodgkin's (e.g., T-cell lymphomas such as peripheral T-cell lymphomas, cutaneous T-cell lymphomas, etc.) lymphoma.

In some embodiments, the invention relates to the treatment of myelomas and/or myelodysplastic syndromes. In some embodiments, the invention relates to treatment of solid tumors. In some such embodiments the invention relates to treatment of solid tumors such as lung, breast, colon, liver, pancreas, renal, prostate, ovarian, and/or brain. In some embodiments, the invention relates to treatment of pancreatic cancer. In some embodiments, the invention relates to treatment of renal cancer. In some embodiments, the invention relates to treatment of prostate cancer. In some embodiments, the invention relates to treatment of sarcomas. In some embodiments, the invention relates to treatment of soft tissue sarcomas. In some embodiments, the invention relates to methods of treating one or more immune-mediated responses and diseases.

For example, in some embodiments, the invention relates to treatment of rejection following transplantation of synthetic or organic grafting materials, cells, organs or tissue to replace all or part of the function of tissues, such as heart, kidney, liver, bone marrow, skin, cornea, vessels, lung, pancreas, intestine, limb, muscle, nerve tissue, duodenum, small-bowel, pancreatic-islet-cell, including xeno-transplants, etc.; treatment of graft-versus-host disease, autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, thyroiditis, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes uveitis, juvenile-onset or recent-onset diabetes mellitus, uveitis, Graves' disease, psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis, vasculitis, auto-antibody mediated diseases, aplastic anemia, Evan's syndrome, autoimmune hemolytic anemia, and the like; and further to treatment of infectious diseases causing aberrant immune response and/or activation, such as traumatic or pathogen induced immune dysregulation, including for example, that which are caused by hepatitis B and C infections, HIV, Staphylococcus aureus infection, viral encephalitis, sepsis, parasitic diseases wherein damage is induced by an inflammatory response (e.g., leprosy). In some embodiments, the invention relates to treatment of graft vs host disease (especially with allogenic cells), rheumatoid arthritis, systemic lupus erythematosus, psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis and/or multiple sclerosis.

Alternatively or additionally, in some embodiments, the invention relates to treatment of an immune response associated with a gene therapy treatment, such as the introduction of foreign genes into autologous cells and expression of the encoded product. In some embodiments, the invention relates to treatment of circulatory diseases, such as arteriosclerosis, atherosclerosis, vasculitis, polyarteritis nodosa and/or myocarditis.

In some embodiments, the invention relates to treatment of any of a variety of neurodegenerative diseases, a non-exhaustive list of which includes:

-   -   I. Disorders characterized by progressive dementia in the         absence of other prominent neurologic signs, such as Alzheimer's         disease; Senile dementia of the Alzheimer type; and Pick's         disease (lobar atrophy);     -   II. Syndromes combining progressive dementia with other         prominent neurologic abnormalities such as A) syndromes         appearing mainly in adults (e.g., Huntington's disease, Multiple         system atrophy combining dementia with ataxia and/or         manifestations of Parkinson's disease, Progressive supranuclear         palsy (Steel-Richardson-Olszewski), diffuse Lewy body disease,         and corticodentatonigral degeneration); and B) syndromes         appearing mainly in children or young adults (e.g.,         Hallervorden-Spatz disease and progressive familial myoclonic         epilepsy);     -   III. Syndromes of gradually developing abnormalities of posture         and movement such as paralysis agitans (Parkinson's disease),         striatonigral degeneration, progressive supranuclear palsy,         torsion dystonia (torsion spasm; dystonia musculorum deformans),         spasmodic torticollis and other dyskinesis, familial tremor, and         Gilles de la Tourette syndrome;     -   IV. Syndromes of progressive ataxia such as cerebellar         degenerations (e.g., cerebellar cortical degeneration and         olivopontocerebellar atrophy (OPCA)); and spinocerebellar         degeneration (Friedreich's ataxia and related disorders);     -   V. Syndromes of central autonomic nervous system failure         (Shy-Drager syndrome);     -   VI. Syndromes of muscular weakness and wasting without sensory         changes (motorneuron disease such as amyotrophic lateral         sclerosis, spinal muscular atrophy (e.g., infantile spinal         muscular atrophy (Werdnig-Hoffman), juvenile spinal muscular         atrophy (Wohlfart-Kugelberg-Welander) and other forms of         familial spinal muscular atrophy), primary lateral sclerosis,         and hereditary spastic paraplegia;     -   VII. Syndromes combining muscular weakness and wasting with         sensory changes (progressive neural muscular atrophy; chronic         familial polyneuropathies) such as peroneal muscular atrophy         (Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy         (Dejerine-Sottas), and miscellaneous forms of chronic         progressive neuropathy;     -   VIII. Syndromes of progressive visual loss such as pigmentary         degeneration of the retina (retinitis pigmentosa), and         hereditary optic atrophy (Leber's disease).

In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, and/or Huntington's disease.

In some embodiments, the invention relates to treatment of disorders, diseases or conditions associated with chromatin remodeling.

In some embodiments, the invention relates to treatment of tumors expressing the Ras oncogene, as discussed more fully in commonly owned co-pending U.S. patent application Ser. No. ______ filed on even date herewith, and entitled “TREATMENT OF RAS-EXPRESSING TUMORS”, a complete copy of which is attached hereto as Exhibit A. As indicated above, Ras-expressing tumors are often more resistant to standard therapies. Ras-expressing tumors are often more resistant to standard therapies. Furthermore, many of the most deadly cancers involve Ras-expressing tumors. For example, 90-95% of pancreatic tumors are Ras-expressing. Similarly, 40-45% of colorectal tumors, 40% of bladder tumors, 15-20% of non small cell lung carcinomas express Ras. Indeed, 10-25% of myelodysplastic syndromes (MDS), which are not themselves cancer but are bone marrow disorders characterized by abnormal cell maturation that typically progress to cancer, also express Ras. There is a profound need for the development of therapies for these and other Ras-expressing diseases and disorders.

DAC Inhibitors

Deacetylase inhibitors, as that term is used herein are compounds which are capable of inhibiting the deacetylation of proteins in vivo, in vitro or both. In many embodiments, the invention relates to HDAC inhibitors, which inhibit the deacetylation of histones. However, those of ordinary skill in the art will appreciate that HDAC inhibitors often have a variety of biological activities, at least some of which may well be independent of histone deacetylase inhibition.

As indicated, DAC inhibitors inhibit the activity of at least one deacetylase. Where the DAC inhibitor is an HDAC inhibitor, an increase in acetylated histones occurs and accumulation of acetylated histones is a suitable biological marker for assessing the activity of HDAC inhibitors. Therefore, procedures which can assay for the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of agents of interest. Analogous assays can determine DAC inhibitory activity

It is understood that agents which can inhibit deacetylase activity (e.g., histone deacetylase activity) typically can also bind to other substrates and as often can inhibit or otherwise regulate other biologically active molecules such as enzymes.

Suitable DAC or HDAC inhibitors according to the present invention include, for example, 1) hydroxamic acid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting histone deacetylase. Examples of such DAC inhibitors include, but are not limited to:

-   A) HYDROXAMIC ACID DERIVATIVES such as Suberoylanilide Hydroxamic     Acid (SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95:3003,     1998); M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Richon et al.,     supra); pyroxamide; CBHA; Trichostatin analogues such as     Trichostatin A (TSA) and Trichostatin C (Koghe et al. Biochem.     Pharmacol. 56:1359, 1998); Salicylihydroxamic Acid (SBHA) (Andrews     et al., International J. Parasitology 30:761, 2000); Azelaic     Bishydroxamic Acid (ABHA) (Andrews et al., supra);     Azelaic-1-Hydroxamate-9-Anilide (AAHA) (Qiu et al., Mol. Biol. Cell     11:2069, 2000); 6-(3-Chlorophenylureido) carpoic Hydroxamic Acid     (3Cl-UCHA), Oxamflatin [(2E)-5-[3-[(phenylsuibnyl-)amino     phenyl]-pent-2-en-4-ynohydroxamic acid (Kim et al. Oncogene, 18:     2461, 1999); A-161906, Scriptaid (Su et al. 2000 Cancer Research,     60:3137, 2000); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews     et al., supra); and MW2996 (Andrews et al., supra). -   B) CYCLIC TETRAPEPTIDES such as Trapoxin A (TPX)-Cyclic Tetrapeptide     (cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amin-o-8-oxo-9,10-epoxy     decanoyl)) (Kijima et al., J Biol. Chem. 268:22429, 1993); FR901228     (FK 228, FR901228, Depsipeptide, Romidepsin) (Nakajima et al., Ex.     Cell Res. 241:12, 1998); FR225497 Cyclic Tetrapeptide (Mori et al.,     PCT Application WO 00/08048, Feb. 17, 2000); Apicidin Cyclic     Tetrapeptide     [cyclo(NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipe-colinyl-L-2-amino-8oxodecanoyl)]     (Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93:13143, 1996);     Apicidin la, Apicidin Ib, Apicidin Ic, Apicidin IIa, and Apicidin     IIb (P. Dulski et al., PCT Application WO 97/11366); CHAP, HC-Toxin     Cyclic Tetrapeptide (Bosch et al., Plant Cell 7:1941, 1995); WF27082     Cyclic Tetrapeptide (PCT Application WO 98/48825); and Chiamydocin     (Bosch et al., supra). -   C) SHORT CHAIN FATTY ACID (SCFA) DERIVATIVES such as: Sodium     Butyrate (Cousens et al., J. Biol. Chem. 254:1716, 1979);     Isovalerate (McBain et al., Biochem. Pharm. 53:1357, 1997); Valerate     (McBain et al., supra); 4 Phenylbutyrate (4-PBA) (Lea and Tulsyan,     Anticancer Research, 15:879, 1995); Phenylbutyrate (PB) (Wang et     al., Cancer Research, 59:2766, 1999); Propionate (McBain et al.,     supra); Butyramide (Lea and Tulsyan, supra); Isobutyramide (Lea and     Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra);     3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et al.,     Cancer Research, 60:749, 2000); Valproic acid and Valproate. -   D) BENZAMIDE DERIVATIVES such as CI-994; MS-275     [N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide]     (Saito et al., Proc. Natl. Acad. Sci. USA 96:4592, 1999; 3′-amino     derivative of MS-27-275 (Saito et al., supra); MGCD0103 (MethylGene;     see FIG. 1), or related compounds (for example, see FIG. 2). -   E) ELECTROPHILIC KETONE DERIVATIVES such as trifluoromethyl ketones     (Frey et al, Bioorganic & Med. Chem. Lett., 12: 3443, 2002; U.S.     Pat. No. 6,511,990) and α-keto amides such as N-methyl-α-ketoamides. -   F) OTHER DAC Inhibitors such as Depudecin (Kwon et al., Proceedings     of the National Academy of Sciences USA, 95:3356, 1998), and     compounds depicted in FIG. 3.

Suitable DAC inhibitors for use in accordance with the present invention particularly include, for example, CRA-024781 (Cetera Genomics), PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCD0103 (MethylGene), MS-275 (Schering AG), romidepsin (Gloucester Pharmaceuticals), and/or SAHA (Alton Pharma/Merck).

In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (I):

wherein

m is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

p and q are independently 1 or 2;

X is O, NH, or NR₈;

-   -   R₁, R₂, and R₃ are independently hydrogen; unsubstituted or         substituted, branched or unbranched, cyclic or acyclic         aliphatic; unsubstituted or substituted, branched or unbranched,         cyclic or acyclic heteroaliphatic; unsubstituted or substituted         aryl; or unsubstituted or substituted heteroaryl;         -   R₄, R₅, R₆, R₇ and R₈ are independently hydrogen; or             substituted or unsubstituted, branched or unbranched, cyclic             or acyclic aliphatic; and pharmaceutically acceptable forms             thereof. In certain embodiments, m is 1. In certain             embodiments, n is 1. In certain embodiments, p is 1. In             certain embodiments, q is 1. In certain embodiments, X is O.             In certain embodiments, R₁, R₂, and R₃ are unsubstituted, or             substituted, branched or unbranched, acyclic aliphatic. In             certain embodiments, R₄, R₅, R₆, and R₇ are all hydrogen.

In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (II):

wherein:

m is 1, 2, 3 or 4;

n is 0, 1, 2 or 3;

-   -   q is 2 or 3;

X is O, NH, or NR₈;

Y is OR₈, or SR₈;

R₂ and R₃ are independently hydrogen; unsubstituted or substituted, branched or unbranched, cyclic or acyclic aliphatic; unsubstituted or substituted, branched or unbranched, cyclic or acyclic heteroaliphatic; unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl;

R₄, R₅, R₆, R₇ and R₈ are independently selected from hydrogen; or substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; and pharmaceutically acceptable forms thereof. In certain embodiments, m is 1. In certain embodiments, n is 1. In certain embodiments, q is 2. In certain embodiments, X is O. In other embodiments, X is NH. In certain embodiments, R₂ and R₃ are unsubstituted or substituted, branched or unbranched, acyclic aliphatic. In certain embodiments, R₄, R₅, R₆, and R₇ are all hydrogen.

In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (III):

wherein

A is a moiety that is cleaved under physiological conditions to yield a thiol group and includes, for example, an aliphatic or aromatic acyl moiety (to form a thioester bond); an aliphatic or aromatic thioxy (to form a disulfide bond); or the like; and pharmaceutically acceptable forms thereof. Such aliphatic or aromatic groups can include a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. A can be, for example, —COR₁, —SC(═O)—O—R₁, or —SR₂. R₁ is independently hydrogen; substituted or unsubstituted amino; substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; substituted or unsubstituted aromatic group; substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. In certain embodiment, R₁ is hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl, benzyl, or bromobenzyl. R₂ is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. In certain embodiments, R₂ is methyl, ethyl, 2-hydroxyethyl, isobutyl, fatty acids, a substituted or unsubstituted benzyl, a substituted or unsubstituted aryl, cysteine, homocysteine, or glutathione.

In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (IV) or (IV′):

wherein R₁, R₂, R₃, and R₄ are the same or different and represent an amino acid side chain moiety, each R₆ is the same or different and represents hydrogen or C₁-C₄ alkyl, and Pr¹ and Pr² are the same or different and represent hydrogen or thiol-protecting group. In certain embodiments, the amino acid side chain moieties are those derived from natural amino acids. In other embodiments, the amino acid side chain moieties are those derived from unnatural amino acids. In certain embodiments, each amino acid side chain is a moiety selected from —H, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, -L-O—C(O)—R′, -L-C(O)—O—R″, -L-A, -L-NR″R″, -L-Het-C(O)—Het-R″, and -L-Het-R″, wherein L is a C₁-C₆ alkylene group, A is phenyl or a 5- or 6-membered heteroaryl group, each R′ is the same or different and represents C₁-C₄ alkyl, each R″ is the same or different and represent H or C₁-C₆ alkyl, each -Het- is the same or different and is a heteroatom spacer selected from —O—, —N(R′″)—, and —S—, and each R′″ is the same of different and represents H or C₁-C₄ alkyl. In certain embodiments, R₆ is —H. In certain embodiments, Pr¹ and Pr² are the same or different and are selected from hydrogen and a protecting group selected from a benzyl group which is optionally substituted by C₁-C₆ alkoxy, C₁-C₆ acyloxy, hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, C₁-C₆ acyloxymethyl, C₁-C₆ alkoxymethyl, tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl, and C₁-C₆ alkylcarbamoyl. In certain embodiments, Pr¹ and Pr² are hydrogen. Various romidepsin derivatives of formula (IV) and (IV′) are disclosed in published PCT application WO 2006/129105, published Dec. 7, 2006; which is incorporated herein by reference.

In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (V):

wherein

-   -   B is a substituted or unsubstituted, saturated or unsaturated         aliphatic group, a substituted or unsubstituted, saturated or         unsaturated alicyclic group, a substituted or unsubstituted         aromatic group, a substituted or unsubstituted heteroaromatic         group, or a substituted or unsubstituted heterocyclic group; R₂₀         is hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino, or         alkyloxy group; R₂₁ and R₂₂ are independently selected from         hydrogen, hydroxyl, a substituted or unsubstituted, saturated or         unsaturated aliphatic group, a substituted or unsubstituted,         saturated or unsaturated alicyclic group, a substituted or         unsubstituted aromatic group, a substituted or unsubstituted         heteroaromatic group, or a substituted or unsubstituted         heterocyclic group. In a particular embodiment of Formula IV,         R₂₀ is a hydroxylamino, hydroxyl, amino, methylamino,         dimethylamino or methyloxy group and B is a C₆-alkyl. In yet         another embodiment of Formula IV, R₂₁ is a hydrogen atom, R₂₂ is         a substituted or unsubstituted phenyl and B is a C₆-alkyl. In         further embodiments of Formula IV, R₂₁ is hydrogen and R₂₂ is an         α-, β-, or γ-pyridine.

Other examples of DAC or HDAC inhibitors can be found in, for example, U.S. Pat. Nos. 5,369,108, issued on Nov. 29, 1994, 5,700,811, issued on Dec. 23, 1997, 5,773,474, issued on Jun. 30, 1998, 5,932,616 issued on Aug. 3, 1999 and 6,511,990, issued Jan. 28, 2003 all to Breslow et al.; U.S. Pat. Nos. 5,055,608, issued on Oct. 8, 1991, 5,175,191, issued on Dec. 29, 1992 and 5,608,108, issued on Mar. 4, 1997 all to Marks et al.; U.S. Provisional Application No. 60/459,826, filed Apr. 1, 2003 in the name of Breslow et al.; as well as, Yoshida, M., et al., Bioassays 17, 423-430 (1995); Saito, A., et al., PNAS USA 96, 4592-4597, (1999); Furamai R. et al., PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et al., Cancer Res. 61(11), 4459-4466 (2001); Su, G. H., et al., Cancer Res. 60, 3137-3142 (2000); Lee, B. I. et al., Cancer Res. 61(3), 931-934; Suzuki, T., et al., J. Med. Chem. 42(15), 3001-3003 (1999); published PCT Application WO 01/18171 published on Mar. 15, 2001 Sloan-Kettering Institute for Cancer Research and The Trustees of Columbia University; published PCT Application WO02/246144 to Hoffmann-La Roche; published PCT Application WO02/22577 to Novartis; published PCT Application WO02/30879 to Prolifix; published PCT Applications WO 01/38322 (published May 31, 2001), WO 01/70675 (published on Sep. 27, 2001) and WO 00/71703 (published on Nov. 30, 2000) all to Methylgene, Inc.; published PCT Application WO 00/21979 published on Oct. 8, 1999 to Fujisawa Pharmaceutical Co., Ltd.; published PCT Application WO 98/40080 published on Mar. 11, 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current patent status of histone deacetylase inhibitors Expert Opin. Ther. Patents (2002) 12(9): 1375-1384 and references cited therein).

Specific non-limiting examples of DAC or HDAC inhibitors are provided in the Table below. It should be noted that the present invention encompasses any compounds which both are structurally similar to the compounds represented below and are capable of inhibiting histone deacetylases.

Title MS-275

DEPSIPEPTIDE

Cl-994

Apicidin

A-161906

Scriptaid

PXD-101

CHAP

LAQ-824

Butyric Acid

Depudecin

Oxumflatin

Trichostatin C

DAC or HDAC inhibitors for use in accordance with the present invention may be prepared by any available means including, for example, synthesis, semi-synthesis, or isolation from a natural source.

DAC or HDAC inhibitors for use in accordance with the present invention may be isolated or purified. For example, synthesized compounds can be separated from a reaction mixture, and natural products can be separated from their natural source, by methods such as column chromatography, high pressure liquid chromatography, and/or recrystallization.

A variety of synthetic methodologies for preparing DAC or HDAC inhibitors are known in the art. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

DAC or HDAC inhibitors for use in accordance with the present invention may be modified as compared with presently known DAC or HDAC inhibitors, for example, by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and may include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

In some embodiments, a DAC (e.g., HDAC) inhibitor for use in accordance with the present invention may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids. The present invention encompasses all such possible isomers, as well as their racemic and optically pure forms to the extent that they have DAC inhibitory activity.

In general, optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981).

In some embodiments, a DAC (e.g., HDAC) inhibitor for use in accordance with the present invention may contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry. The present invention encompasses both E and Z geometric isomers or cis- and trans-isomers to the extent that they have DAC inhibitory activity. The present invention likewise encompasses all tautomeric forms that have DAC inhibitory activity. In general, where a chemical structure is presented, the configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states or it is otherwise clear from context; thus a carbon-carbon double bond or carbon-heteroatom double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.

DAC inhibitors (e.g., HDAC inhibitors) are particularly useful in the treatment of neoplasms in vivo. However, they may also be used in vitro for research or clinical purposes (e.g., determining the susceptibility of a patient's disease to a particular DAC inhibitor). In certain embodiments, the neoplasm is a benign neoplasm. In other embodiments, the neoplasm is a malignant neoplasm. Any cancer may be treated using a DAC inhibitor alone or in combination with another pharmaceutical agent.

In certain embodiments, the malignancy is a hematological malignancy. Manifestations can include circulating malignant cells as well as malignant masses. Hematological malignancies are types of cancers that affect the blood, bone marrow, and/or lymph nodes. Examples of hematological malignancies that may be treated using romidepsin include, but are not limited to: acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), multiple myeloma, and myelodysplastic syndromes. In certain embodiments, the inventive combination is used to treat multiple myeloma. In certain particular embodiments, the cancer is relapsed and/or refractory multiple myeloma. In other embodiments, the inventive combination is used to treat chromic lymphocytic leukemia (CLL). In certain particular embodiments, the cancer is relapsed and/or refractory CLL. In other embodiments, the inventive combination is used to treat chromic myelogenous leukemia (CML). In certain embodiments, the inventive combination is used to treat acute lymphoblastic leukemia (ALL). In certain embodiments, the inventive combination is used to treat acute myelogenous leukemia (AML). In certain embodiments, the cancer is cutaneous T-cell lymphoma (CTCL). In other embodiments, the cancer is peripheral T-cell lymphoma (PTCL). In certain embodiments, the cancer is a myelodysplastic syndrome.

Other cancers besides hematological malignancies may also be treated using DAC inhibitors. In certain embodiments, the cancer is a solid tumor.

Exemplary cancers that may be treated using DAC inhibitor therapy, including combination therapy, include colon cancer, lung cancer, bone cancer, pancreatic cancer, stomach cancer, esophageal cancer, skin cancer, brain cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, testicular cancer, prostate cancer, bladder cancer, kidney cancer, neuroendocrine cancer, etc.

In certain embodiments, a DAC inhibitor is used to treat pancreatic cancer. In certain embodiments, a DAC inhibitor is used to treat prostate cancer. In certain specific embodiments, the prostate cancer is hormone refractory prostate cancer. In certain embodiments, a DAC inhibitor is administered in combination with one or more additional therapeutic agents, e.g., another cytotoxic agent. Exemplary cytotoxic agents that may be administered in combination with a DAC inhibitor include gemcitabine, decitabine, and flavopiridol. In other embodiments, a DAC inhibitor is administered in combination with an anti-inflammatory agent such as aspirin, ibuprofen, acetaminophen, etc., pain reliever, anti-nausea medication, or anti-pyretic. In certain other embodiments, a DAC inhibitor is administered in combination with a steroidal agent (e.g., dexamethasone). In certain embodiments, a DAC inhibitor is administered in combination with an agent to treat gastrointestinal disturbances such as nausea, vomiting, and diarrhea. These additional agents may include anti-emetics, anti-diarrheals, fluid replacement, electrolyte replacement, etc. In other embodiments, a DAC inhibitor is administered in combination with electrolyte replacement or supplementation such as potassium, magnesium, and calcium, in particular, potassium and magnesium. In certain embodiments, a DAC inhibitor is administered in combination an anti-arrhythmic agent. In certain embodiments, a DAC inhibitor is administered in combination with a platelet booster, for example, an agent that increases the production of platelets. In certain embodiments, a DAC inhibitor is administered in combination with an agent to boost the production of blood cells such as erythropoietin. In certain embodiments, a DAC inhibitor is administered in combination with an agent to prevent hyperglycemia. In certain embodiments, a DAC inhibitor is not administered with another HDAC or DAC inhibitor.

Combination Therapy

The present invention demonstrates the particular utility of administering a combination of a DAC inhibitor and gemcitabine. In some particular embodiments of the present invention, the DAC inhibitor is romidepsin (aka depsipeptide, FK228, FR901228). In other particular embodiments, the DAC inhibitor is selected from the group consisting of CRA-024781 (Cetera Genomics), phenylbutarate, PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCD0103 (MethylGene), MS-275 (Schering AG), romidepsin (Gloucester Pharmaceuticals), SAHA (Alton Pharma/Merck), and combinations thereof. In some particular embodiments of the present invention, the DAC inhibitor is romidepsin (aka depsipeptide, FK228, FR901228). In some particular embodiments, the DAC inhibitor is SAHA. In some particular embodiments, the DAC inhibitor is phenylbutyrate. In some particular embodiments, the DAC inhibitor comprises a combination of DAC inhibitors.

The present invention demonstrates the particular utility of administering a combination of a DAC inhibitor and gemcitabine. Without wishing to be bound by any particular theory, the inventors note that such a combination may increase apoptosis in recipients.

As will be appreciated by those of skill in the art, and as is otherwise addressed herein, either or both of the DAC inhibitor and gemcitabine may be provided in any useful form including, for example, as a salt, ester, active metabolite, prodrug, etc. Similarly, either or both agents (or salts, esters, or prodrugs thereof) may be provided as a pure isomer stereoisomer or as a combination of stereoisomers, including a racemic combination, so long as relevant activity is present. Comparably, either or both agents (or salts, esters or prodrugs thereof) may be provided in crystalline form, whether a pure polymorph or a combination of polymorphs, or in amorphous form, so long as relevant activity is present.

As addressed above, combination therapy of DAC inhibitors and gemcitabine will typically involve administration of multiple individual doses spaced out in time. In some embodiments, individual DAC inhibitor doses and gemcitabine doses will be administered together, according to the same schedule. In other embodiments, DAC inhibitor doses and gemcitabine doses will be administered according to different schedules.

The total daily dose of any particular active agent administered to a human or other animal in single or in divided doses in accordance with the present invention can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. In certain embodiments, about 10-100 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 100-500 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 250-500 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 500-750 mg of the compound is administered per day in single or multiple doses.

In the treatment of neoplasms such as cancer in a subject, a DAC inhibitor is typically dosed at 1-30 mg/m². In certain embodiments, a DAC inhibitor is dosed at 1-15 mg/m². In certain embodiments, a DAC inhibitor is dosed at 5-15 mg/m². In certain particular embodiments, a DAC inhibitor is dosed at 4, 6, 8, 10, 12, 14, 16, 18, or 20 mg/m². A DAC inhibitor is typically administered in a 28 day cycle with the agent being administered on days 1, 8 and 15. In certain embodiments, the DAC is administered on days 1 and 15 with day 8 being skipped. As would be appreciated by one of skill in the art, the dosage and timing of administration of the dosage of the DAC inhibitor may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of DAC inhibitor administered.

Typical dosing schedules have been established for certain exemplary DAC inhibitors (e.g., HDAC inhibitors). For example, SAHA is commonly administered within a range of about 300-400 mg daily orally; PXD101 is commonly administered within a range of about up to 2000 mg/m²/day intravenously (e.g., on days 1 to 5 of a 21 day cycle), and may possibly be administered orally; MGCD0103 is commonly administered at doses up to about 27 mg/m² given orally (e.g., daily for about 14 days); LBH589 is commonly administered at doses up to about 14 mg/m² as an intravenous infusion (e.g., on days 1-7 of a 21 day cycle); MS-275 is commonly administered within a dose range of about 2-12 mg/m² intravenously (e.g., every 14 days).

In the treatment of neoplasms such as cancer in a subject, romidepsin is typically dosed at 1-28 mg/m². In certain embodiments, romidepsin is dosed at 1-15 mg/m². In certain embodiments, romidepsin is dosed at 5-14 mg/m². In certain particular embodiments, romdiepsin is dosed at 8, 10, 12, or 14 mg/m². Romidepsin is typically administered in a 28 day cycle with romidepsin being administered on days 1, 8 and 15. In certain embodiments, romidepsin is administered on days 1 and 15 with day 8 being skipped.

Acceptable dosing schedules have also been established for gemcitabine for at least pancreatic, non-small cell lung, breast, and ovarian cancers. For example, for pancreatic cancer, gemcitabine is typically administered by intravenous infusion at a dose of 1000 mg/m² over 30 minutes once weekly for up to 7 weeks (or until toxicity necessitates reducing or holding a dose), followed by a week of rest from treatment. Subsequent cycles typically consist of infusions once weekly for 3 consecutive weeks out of every 4 weeks.

For non-small cell lung cancer, where it is typically given in combination, gemcitabine is often administered either on a 4-week schedule that involves intravenous dosing at 1000 mg/m² over 30 minutes on Days 1, 8, and 15 of each 28-day cycle, or on a 3-week schedule, where it is administered intravenously at 1250 mg/m² over 30 minutes on Days 1 and 8 of each 21-day cycle.

For breast cancer, where it is typically also given in combination, gemcitabine is often administered intravenously at a dose of 1250 mg/m² over 30 minutes on Days 1 and 8 of each 21-day cycle.

For ovarian cancer, where it is typically also given in combination, gemcitabine is often administered intravenously at a dose of 1000 mg/m² over 30 minutes on Days 1 and 8 of each 21-day cycle.

As would be appreciated by one of skill in the art, the dosage and timing of administration of any particular DAC inhibitor or gemcitabine dose, or the dosage amount and schedule generally may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of one or the other agent, or of both agents, being administered.

Moreover, those of ordinary skill in the art will readily appreciate that the dosage schedule (i.e., amount and timing of individual doses) by which any particular DAC inhibitor is administered may be different for inventive combination therapy with gemcitabine than it is alone. Comparably, the dosage schedule for gemcitabine may be different according to inventive combination therapy regimens than would be utilized in gemcitabine monotherapy (even for the same disorder, disease or condition).

To give but one example, in some embodiments, a DAC inhibitor (e.g., romidepsin) and gemcitabine are each dosed on days 1 and 15 of a 28 day cycle. Those of ordinary skill in the art will appreciate that any of a variety of other dosing regimens are within the scope of the invention. Commonly, dosing is adjusted based on a patient's response to therapy, and particularly to development of side effects.

In some embodiments of the present invention, inventive combination therapy with one or more DAC inhibitors and gemcitabine is further combined with administration of one or more other agents.

In some embodiments, subjects receiving inventive combination therapy with one or more DAC inhibitors and gemcitabine further receive electrolyte supplementation for example as is described in co-pending U.S. Provisional Patent application Ser. No. 60/909,780 entitled “DEACETYLASE INHIBITOR THERAPY”, filed Apr. 3, 2007.

For example, as described in that application, an individual with a potassium serum concentration below about 3.5 mmol/L (3.5 mEq/L) and/or a serum magnesium concentration below about 0.8 mml/L (1.95 mEq/L) suffers an increased risk of developing cardiac repolarization effects and/or dysrhythmias.

Serum concentrations of potassium are generally considered to be “normal” when they are within the range of about 3.5-5.5 mEq/L or about 3.5-5.0 mEq/L. According to the present invention, it is often desirable to ensure that an individuals' serum potassium concentration is within this range prior to (and/or during) administration of DAC inhibitor therapy.

Serum concentrations of magnesium are generally considered to be “normal” when they are within the range of about 1.5-2.5 mEq/L or about 1.5-2.2 mEq/L or about 1.25-2.5 mEq/L or about 1.25-2.2 mEq/L. According to the present invention, it is often desirable to ensure that an individual's serum magnesium concentration is within this range prior to (and/or during) administration of DAC inhibitor therapy.

In some embodiments of the invention, an individual's serum potassium and/or magnesium concentration(s) is/are at the high end of the normal range prior to (and/or during) administration of DAC inhibitor therapy. For example, in some embodiments, an individual's serum potassium concentration is at least about 3.8, 3.9, 4.0 mEq/L, or more prior to and/or during administration of DAC inhibitor therapy. In some embodiments, care is taken not to increase serum potassium concentration above about 5.0, 5.2, or 5.5 mEq/L. In some embodiments, an individual's serum magnesium concentration is at least about 1.9 mEq/L or more prior to and/or during administration of DAC inhibitor therapy. In some embodiments, care is taken not to increase magnesium concentration above about 2.5 mEq/L.

In some embodiments of the present invention, an individual's serum potassium concentration is at least about 3.5 mEq (in some embodiments at least about 3.8, 3.9, 4.0 mEq/L or above) and the individual's serum magnesium concentration is at least about 1.85 mEq/L (in some embodiments at least about 1.25, 1.35, 1.45, 1.55, 1.65, 1.75, 1.85, 1.95, etc) prior to and/or during administration of DAC inhibitor therapy.

In some embodiments of the invention, electrolyte levels (e.g., potassium and/or magnesium levels, optionally calcium levels) are assessed more than once during the course of DAC inhibitor therapy; in some embodiments, different assessments are separated by a regular interval (e.g., 0.5 days or less, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc.). In some embodiments, electrolyte levels are assessed prior to each administration of DAC inhibitor.

Pharmaceutical Compositions

DAC inhibitors and/or gemcitabine for use in accordance with the present invention are often administered as pharmaceutical compositions comprising amounts of DAC inhibitor and gemcitabine, respectively, that are useful in inventive combination therapy (which amounts may be different from, including less than, amounts required for either agent to be effective alone). In some embodiments, a DAC inhibitor and gemcitabine are present together in a single pharmaceutical composition; in some embodiments these agents are provided in separate pharmaceutical compositions.

In some embodiments, inventive pharmaceutical compositions are prepared in unit dosage forms. In general, a pharmaceutical composition of the present invention includes one or more active agents (i.e., one or more DAC inhibitors and/or gemcitabine) formulated with one or more pharmaceutically acceptable carriers or excipients.

In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening, flavoring and perfuming agents; preservatives and antioxidants; and combinations thereof. In some embodiments, the pH of the ultimate pharmaceutical formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.

Pharmaceutical compositions of this invention may be administered can be administered by any appropriate means including, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In many embodiments, pharmaceutical compositions are administered orally or by injection in accordance with the present invention.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), liquid dosage forms of pharmaceutical compositions may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from a site of subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the active agents with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent(s) is/are typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents, permeation enhancers, and/or other agents to enhance absorption of the active agent(s).

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

In certain embodiments, oral dosage forms are prepared with coatings or by other means to control release of active agent (e.g., DAC inhibitor and/or gemcitabine) over time and/or location within the gastrointestinal tract. A variety of strategies to achieve such controlled (or extended) release are well known in the art, and are within the scope of the present invention.

Dosage forms for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In general, such preparations are prepared by admixing active agent(s) under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.

Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

Ointments, pastes, creams and gels may contain, in addition to active agent(s), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to active agent(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

Transdermal patches have often can provide controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

For pulmonary delivery, active agent(s) is/are formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system. Solid or liquid particulate forms of the active agent(s) prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, particularly aerosolized antibiotics, is known in the art (see, for example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat. No. 5,508,269 to Smith et al., and WO 98/43,650 by Montgomery, all of which are incorporated herein by reference). A discussion of pulmonary delivery of antibiotics is also found in U.S. Pat. No. 6,014,969, incorporated herein by reference.

Pharmaceutical compositions for use in accordance with the present invention can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, for example with a dosage ranging from about 0.1 to about 500 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug.

The methods herein contemplate administration of an effective amount of active agent or pharmaceutical composition sufficient for a desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.

The amount of any particular active agent that may be combined with pharmaceutically acceptable excipients or carriers to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations may contain from about 20% to about 80% active compound. For romidepsin, preparations may commonly contain about 20-50%, 25-45%, 30-40%, or approximately 32%, 33%, 34%, or 35% active compound; for gemcitabine, the compound is typically provided in 200 mg or 1 gram vials as a lyophilized powder. Drug product is reconstituted with either 5 ml (for the 200 mg vial) or 25 ml (for the 1 g vial) using sodium chloride for injection. Both dilutions give a 38 mg/ml solution (including displacement volume). This solution can be diluted down to 0.1 mg/ml-0.4 mg/ml for administration.

Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

When pharmaceutical compositions contain two or more active agents, it is generally the case that each agent is present at dosage levels of between about 1 to 100%, for example about 5 to 95%, of the level normally administered in a monotherapy regimen.

Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one of ordinary skill in the art. All publications, patents, published patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety. The embodiments of the invention should not be deemed to be mutually exclusive and can be combined.

Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one of ordinary skill in the art. All publications, patents, published patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety. The embodiments of the invention should not be deemed to be mutually exclusive and can be combined.

EXEMPLIFICATION

The present invention will be better understood in connection with the following Examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.

Example 1 Depsipeptide (FK228) Alone and in Combination with Gemcitabine in In Vivo Mouse Xenograft Model of Ras-Expressing Pancreatic Tumor

The present Example demonstrates that both depsipeptide (FK228; FR901228; romidepsin) and gemcitabine can effectively inhibit tumor growth in a mouse xenograft model, and further demonstrates a surprising synergistic effect of the combination.

Panc-1, obtained from the ATCC, is a pancreas tumor cell line (oncogenic K-ras) originating from a 56 year old Caucasian male. In this study, female nude mice were implanted subcutaneously (SC) by trocar with Panc-1 tumor fragments harvested from SC growing tumors in nude mice hosts. When tumors reaches approximately 140 mm³, animals were pair matched by tumor size into treatment and control groups (N=9 mice per group) (FIG. 4). The day of treatment initiation was specified as Day 1. Vehicle control and FK228 were administered intravenously on a Q4D×3 schedule (Days 1, 5, and 9). Gemcitabine was administered by an intrperitoneal injection on a Q3D×4 schedule (Days 1, 4, 7, 10). Tumors were measured by Vernier calipers twice weekly.

Treatment with depsipeptide (FK228, FR901228, romidepsin) and gemcitabine as single agents both resulted in consistently smaller tumors than vehicle control treated animals, with tumor growth inhibitions of 26% and 50%, respectively.

In addition, a potential synergistic effect of depsipeptide and gemcitabine was observed in combination with a significant tumor growth inhibition of 101%. Three animals in the combination group exhibited evidence of tumor regression. These results indicate depsipeptide has clear antitumor activity against the Panc-1 human pancreas tumors in an in vivo xenograft model. Furthermore, depsipeptide has the potential to synergize with other approved chemotherapeutics, and specifically with gemcitabine. The effects of this synergy, including tumor regression, are particularly significant given the known aggressiveness of pancreatic tumors, and their susceptibility to developing resistance. The present invention demonstrates tumor regression after dosing with a combination of romidepsin and gemcitabine. Note that no regression was observed with gemcitabine alone, the current standard therapy for pancreatic tumors, yet regression was observed with the combination.

We note that no synergistic effect was observed with the combination of romidepsin and gemcitabine in another cell line (Bx-PC-3) that had normal Ras.

Example 2 Combination of FK228 and Gemcitabine is More Effective than Either Agent Alone in a Ras-Transformed Pancreatic Adenocarcinoma Model

The present Example demonstrates that the combination of FK228 and gemcitabine is more effective than either agent alone in a pancreatic adenocarcinoma model.

Abstract:

To examine activity and mechanism of FK228, antitumor efficacy was tested in PANC-1 pancreatic adenocarcinoma model representing transformed Ras, either as a single agent or in combination with gemcitabine. Following PANC-1 study completion, tumor and sera were obtained from:

-   -   the vehicle control;     -   FK228 dosed at 5 mg/kg once every four days for three treatments         (Q4D×3);

Gemcitabine at 80 mg/kg (Q3D×4); and

-   -   the drug combination.         Expression of c-Myc, acetylated histones 3 and 4, and p21^(waf)1         was compared between control and FK228 groups by immunoblotting         and was quantified following actin normalization. Serum levels         of putative tumor products b-FGF and MMP-2 were quantified by         human-specific ELISA.

Highly significant (p<0.0001) downregulation of c-Myc was observed in all treatment groups, most dramatically in the combination group. Acetylated histone 3 levels were not affected in FK228 alone or in combination with gemcitabine. Upregulation of acetylated histone 4 by the drug combination was highly significant. Treatment with gemcitabine alone significantly (p<0.05) downregulated p21^(waf); however, this effect was not reported in combination groups.

These results suggest activity of FK228 is Ras-transformed malignancies and demonstrate combinatorial effects with gemcitabine at least in pancreatic adenocarcinoma. Surprising long-term effects of FK228 in combination with gemcitabine on c-Myc and acetylated histone 4 might suggest tumor phenotypic changes consistent with downregulation of HDAC activity.

Materials and Methods

Specimen Collection: FK228 antitumor efficacy was tested in PANC-1 pancreatic adenocarcinoma model representing transformed Ras, either as a single agent or in combination with gemcitabine. Tumor xenograft tissue and serum specimens were obtained from in vivo studies performed by the Preclinical Research Laboratory at the completion of the experiments. The following tumor and sera specimens were obtained:

-   -   Vehicle control (n−9)     -   FK228 at 5 mg/kg once every four days for three treatments         (Q4D×3) (n=9)     -   Gemcitabine at 80 mg/kg (Q3D×4) (n=9)     -   FK228 at 5 mg/kg plus gemcitabine at 80 mg/kg (Q3D×4) (n=6)         Tumors were dissected from the animals, rinsed in cold phosphate         buffered saline, and snap frozen in liquid nitrogen. Serum was         obtained from whole blood and stored frozen at −70° C.

Tissue Biomarkers: Tumor levels of acetylated histone-4, histone-4, c-Myc, p21waf and β-actin were quantified by immunoblotting as described. Briefly, frozen tissue was pulverized under liquid nitrogen and homogenized in hypotonic lysis buffer. Small aliquots of the extracts were used for analysis of protein concentration by micro-BCA assay with bovine serum albumin as a protein standard/An equal amount of extracts containing about 20-50 μg protein was electrophoresed in SDS polyacrylamide gels. Proteins were transferred to ImmunoBlot PVDF membrane and were probed with appropriate primary and secondary antibodies. The chemiluminescence signal was captured by autoradiography, quantified by densitometry and expressed as a ratio of actin in each sample lane. For each biomarker, means and standard errors were calculated in each treatment group. The data were analyzed by two-sided t-tests to determine if measured end points are significantly affected by drug treatment.

Serum Biomarkers: Serum levels of b-FGF and VEGF were quantified by ELISA using human-specific kits from R&D Systems, Minneapolis, Minn., according to supplier's instructions. All assays were performed in duplicate. For each biomarker, means and standard errors were calculated in each treatment group. The data were analyzed by two-tailed Student t-tests to determine if measured endpoints are significantly affected by drug treatment.

Results

The results of western blot detection of acetylated histone 3, histone 4, c-Myc, p21^(waf) and a housekeeping gene product β-actin as an internal control are shown in FIG. 4. Uniform expression of β-actin was noted in all samples. Following quantitative analysis of biomarker levels in each sample, the results were normalized for b-actin and expressed as percentages of untreated controls. Group averages were compared by t-test.

When compared with the controls, the expression of c-Myc was inhibited in all groups. The extent of inhibition (50%) was similar in the gemcitabine and FK228 monotherapy treatment groups and greater (60%) in the combination group. The inhibition of c-Myc expression was highly significant (p<0.0001) in all cases.

Acetylation of histone 3 was significantly inhibited by gemcitabine, but not affected by FK228 alone or in combination with gemcitabine.

The levels of acetylated histone 4 were on the control level in the gemcitabine group. FK228 treatment induced over 2-fold increase of acetylated histone 4, but in comparison with the control group the increase was not significant. On the other hand, over 3-fold up-regulation of acetylated histone 4 by the drug combination was highly significant (p=0.00003).

Treatment with gemcitabine alone significantly (p<0.05) down-regulated p21^(waf); however, this effect was not observed in the combination groups.

Quantitative analysis of b-FGF and VEGF in serum was also performed. The levels of b-FGF were highly variable but not significantly different in any treatment groups in comparison with the controls. VEGF was under the detection limits of the assay.

The effects of FK228 on expression of c-Myc and acetylated histone 4 are unexpected considering that these endpoints were assessed at the end of a long-term in vivo treatment with the drug. Historically, the effects of DAC inhibitors such as FK228 on target gene or protein expression were assessed in a time scale of hours (not days) following drug treatment. For example, a study on the effects of FK228 on tumor growth and expression of p21 and c-myc genes in vivo over a period of 2 to 24 hours demonstrated induction of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive to FK228, while opposite effects on p21 and c-myc mRNA were seen in tumor xenograft less sensitive to FK228.

Myc genes are key regulators of cell proliferation, and their deregulation contributes to the genesis of most human tumors. Transcriptional regulation by Myc-family proteins includes recruitment of HDACs in tumors, some of which exhibit dependence (addition) to c-myc. Even a brief inhibition of c-myc expression may be sufficient to completely stop tumor growth and induce regression of tumors. It is conceivable that biological activity of FK228 could be partly due to inhibition of c-myc and other genes under its control, including HDACs.

In conclusion, these results demonstrate at least additive combinatorial effects with gemcitabine on the expression of c-Myc and acetylation of histone 4 in pancreatic adenocarcinoma. Surprising effects, of FK228 in combination with gemcitabine might suggest tumor phenotypic changes consistent with downregulation of HDAC activity. Specifically, weeks after the end of treatment, the cells are phenotypically different from those that were initially injected, suggesting some form of cellular transformation, possible to a less aggressive phenotype.

EQUIVALENTS

The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

To give but a few examples, in the claims articles such as “a”, “an”, and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. In addition, the invention encompasses compositions made according to any, of the methods for preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included unless otherwise indicated. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention can be excluded from any one or more claims. For example, in certain embodiments of the invention the biologically active agent is not an anti-proliferative agent. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein. 

1. A method comprising steps of: administering to a subject suffering from or susceptible to a cell proliferative disorder, combination therapy of a DAC inhibitor and gemcitabine.
 2. The method of claim 1 wherein the cell proliferative disorder involves a tumor.
 3. The method of claim 2, wherein the tumor is a Ras-expressing tumor.
 4. The method of claim 2, wherein the tumor is a pancreatic tumor.
 5. The method of claim 1, wherein the DAC inhibitor is romidepsin.
 6. The method of claim 1 further comprising administering electrolyte supplementation.
 7. The method of claim 1, wherein the cell proliferative disorder is cutaneous T-cell lymphoma.
 8. The method of claim 1, wherein the cell proliferative disorder is peripheral T-cell lymphoma.
 9. The method of claim 1, wherein the cell proliferative disorder is a hematological malignancy. 